Modifications for Antisense Compounds

ABSTRACT

The invention pertains to modifications for antisense oligonucleotides, wherein the modifications are used to improve stability and provide protection from nuclease degradation. The modifications could also be incorporated into double-stranded nucleic acids, such as synthetic siRNAs and miRNAs.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from U.S. ProvisionalApplication No. 61/380,586, filed Sep. 7, 2010, the disclosure of whichis incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part by Small Business InnovationResearch (SBIR) Grants Nos. 5R44GM085863-02 and 5R44GM085863-03 from theNational Institute of General Medical Sciences of the NationalInstitutes of Health (NIH). The government may have certain rights inthis invention.

FIELD OF THE INVENTION

This invention pertains to modifications for antisense oligonucleotides,wherein the modifications are used to improve stability and provideprotection from nuclease degradation.

BACKGROUND OF THE INVENTION

Antisense oligonucleotides (ASOs) are synthetic nucleic acids that bindto a complementary target and suppress function of that target.Typically ASOs are used to reduce or alter expression of RNA targets,particularly messenger RNA (mRNA) or microRNA (miRNA) species. As ageneral principle, ASOs can suppress gene expression via two differentmechanisms of action, including: 1) by steric blocking, wherein the ASOtightly binds the target nucleic acid and inactivates that species,preventing its participation in cellular biology, or 2) by triggeringdegradation, wherein the ASO binds the target and leads to activation ofa cellular nuclease that degrades the targeted nucleic acid species. Oneclass of “target degrading” ASOs are “RNase H active”; formation ofheteroduplex nucleic acids by hybridization of the target RNA with aDNA-containing “RNase H active” ASO forms a substrate for the enzymeRNase H. RNase H degrades the RNA portion of the heteroduplex molecule,thereby reducing expression of that species. Degradation of the targetRNA releases the ASO, which is not degraded, which is then free torecycle and can bind another RNA target of the same sequence. For anoverview of antisense strategies, oligonucleotide design, and chemicalmodifications, see Kurreck, 2003, Eur. I Biochem., 270(8): 1628-44.

Unmodified DNA oligonucleotides have a half-life of minutes whenincubated in human serum. Therefore, unmodified DNA oligonucleotideshave limited utility as ASOs. The primary nuclease present in serum hasa 3′-exonuclease activity (Eder et al., 1991, Antisense Res. Dev. 1(2):141-51). Once an ASO gains access to the intracellular compartment, itis susceptible to endonuclease degradation. Historically, the firstfunctional ASOs to gain widespread use comprised DNA modified withphosphorothioate groups (PS). PS modification of the internucleotidelinkages confers nuclease resistance, making the ASOs more stable bothin serum and in cells. As an added benefit, the PS modification alsoincreases binding of the ASO to serum proteins, such as albumin, whichdecreases the rate of renal excretion following intravenous injection,thereby improving pharmacokinetics and improving functional performance(Geary et al., 2001, Curr. Opin. Investig. Drugs, 2(4): 562-73).However, PS-modified ASOs are limited to a 1-3 day half-life in tissue,and the PS modifications reduce the binding affinity of the ASO for thetarget RNA, which can decrease potency (Stein et al., 1988, NucleicAcids Res. 16(8): 3209-21).

The PS modification is unique in that it confers nuclease stability yetstill permits formation of a heteroduplex with RNA that is a substratefor RNase H action. Most other modifications that confer nucleaseresistance, such as methyl phosphonates or phosphoramidates, aremodifications that do not form heteroduplexes that are RNase Hsubstrates when hybridized to a target mRNA. Improved potency could beobtained using compounds that were both nuclease resistant and showedhigher affinity to the target RNA yet retain the ability to activateRNase H mediated degradation pathways.

Further design improvements were implemented to increase affinity forthe target RNA while still maintaining nuclease resistance (see Walderet al., U.S. Pat. No. 6,197,944 for designs containing 3′-modificationswith a region containing unmodified residues with phosphodiesterlinkages; see also European Patent No. 0618925 for “Gapmer” compoundshaving 2′-methoxyethylriboses (MOE's) providing 2′-modified ‘wings’ atthe 3′ and 5′ ends flanking a central 2′-deoxy gap region). The newstrategy allows for chimeric molecules that have distinct functionaldomains. For example, a single ASO can contain a domain that confersboth increased nuclease stability and increased binding affinity butitself does not form an RNase H active substrate; a second domain in thesame ASO can be RNase H activating. Having both functional domains in asingle molecule improves performance and functional potency in antisenseapplications. One successful strategy is to build the ASO from differentchemical groups with a domain on each end intended to confer increasedbinding affinity and increased nuclease resistance that flank a centraldomain comprising different modifications which provides for RNase Hactivation. This so-called “end blocked” or “gapmer” design is the basisfor the improved function “second generation” ASOs. Compounds of thisdesign are typically significantly more potent as gene knockdown agentsthan the “first generation” PS-DNA ASOs.

Typically ASOs that function using steric blocking mechanisms of actionshow higher potency when made to maximize binding affinity. This can beaccomplished using chemical modifications that increase bindingaffinity, such as many of the 2′-ribose modifications discussed herein,minor groove binders, or the internal non-base modifiers of the presentinvention. Alternatively, increased binding affinity can be achieved byusing longer sequences. However, some targets are short, such as miRNAs,which are typically only 20-24 bases long. In this case, making ASOslonger to increase binding affinity is not possible. Further, shortsynthetic oligonucleotides gain access into cells more efficiently thanlong oligonucleotides, making it desirable to employ short sequenceswith modifications that increase binding affinity (see, e.g., Straarupet al., 2010, Nucleic Acids Res. 38(20): 7100-11). The chemicalmodification and methods of the present invention enable synthesis ofrelatively short ASOs having increased binding affinity that showimproved functional performance.

ASO modifications that improve both binding affinity and nucleaseresistance typically are modified nucleosides that are costly tomanufacture. Examples of modified nucleosides include locked nucleicacids (LNA), wherein a methyl bridge connects the 2′-oxygen and the4′-carbon, locking the ribose in an A-form conformation; variations ofLNA are also available, such as ethylene-bridged nucleic acids (ENA)that contain an additional methyl group, amino-LNA and thio-LNA.Additionally, other 2′-modifications, such as 2′-O-methoxyethyl (MOE) or2′-fluoro (2′-F), can also be incorporated into ASOs. Some modificationsdecrease stability, and some can have negative effects such as toxicity(see Swayze et al., 2007, Nucleic Acids Res. 35(2): 687-700).

The present invention provides for non-nucleotide modifying groups thatcan be inserted between bases in an ASO to improve nuclease resistanceand binding affinity, thereby increasing potency. The novelmodifications of the present invention can be employed with previouslydescribed chemical modifications (such as PS internucleotide linkages,LNA bases, MOE bases, etc.) and with naturally occurring nucleic acidbuilding blocks, such as DNA or 2′-O-Methyl RNA (2′OMe), which areinexpensive and non-toxic. These and other advantages of the invention,as well as additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides non-nucleotide modifications for antisenseoligonucleotides, wherein the modifications are used to increase bindingaffinity and provide protection from nuclease degradation.

The invention also provides an antisense oligonucleotide comprising atleast one modification that is incorporated between two bases of theantisense oligonucleotide, wherein the modification increases bindingaffinity and nuclease resistance of the antisense oligonucleotide. Inone embodiment, the antisense oligonucleotide comprises at least onemodification that is located within three bases of a terminalnucleotide. In another embodiment, the antisense oligonucleotidecomprises at least one modification that is located between a terminalbase and a penultimate base of either the 3′- or the 5′-end of theoligonucleotide. In a further embodiment, the antisense oligonucleotidecomprises a modification between the terminal base and the penultimatebase of both the 3′- and the 5′-ends of the antisense oligonucleotide.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated between two bases of theantisense oligonucleotide, wherein the modification increases bindingaffinity and nuclease resistance of the antisense oligonucleotide, andwherein the modification is a napthylene-azo compound.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated between two bases of theantisense oligonucleotide, wherein the modification increases bindingaffinity and nuclease resistance of the antisense oligonucleotide, andwherein the modification has the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁-R₅ areindependently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, an electron donatinggroup, or an attachment point for a ligand; and X is a nitrogen orcarbon atom, wherein if X is a carbon atom, the fourth substituentattached to the carbon atom can be hydrogen or a C1-C8 alkyl group.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated between two bases of theantisense oligonucleotide, wherein the modification increases bindingaffinity and nuclease resistance of the antisense oligonucleotide, andwherein the modification has the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁, R₂, R₄,R₅ are independently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, or an electrondonating group; R₆, R₇, R₉-R₁₂ are independently a hydrogen, alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawinggroup, or an electron donating group; R₈ is a hydrogen, alkyl, alkynyl,alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substitutedaryl, cycloalkyl, alkylaryl, alkoxy, or an electron withdrawing group;and X is a nitrogen or carbon atom, wherein if X is a carbon atom, thefourth substituent attached to the carbon atom can be hydrogen or aC1-C8 alkyl group. In one embodiment, R₈ is NO₂.

The invention further provides an antisense oligonucleotide comprisingat least one modification that is incorporated between two bases of theantisense oligonucleotide, wherein the modification increases bindingaffinity and nuclease resistance of the antisense oligonucleotide, andwherein the modification has the structure:

The antisense oligonucleotides of the invention can include natural,non-natural, or modified bases known in the art. The antisenseoligonucleotides of the invention can also include, typically but notnecessarily on the 3′ or 5′ ends of the oligonucleotide, additionalmodifications such as minor groove binders, spacers, labels, or othernon-base entities. In one embodiment, the antisense oligonucleotidefurther comprises 2′-O-methyl RNA, and optionally comprises at least onenapthylene-azo compound. In another embodiment, the antisenseoligonucleotide further comprises phosphorothioate linkages. In afurther embodiment, the antisense oligonucleotide comprises a region ofbases linked through phosphodiester bonds, wherein the region is flankedat one or both ends by regions containing phosphorothioate linkages.

The invention further provides an antisense oligonucleotide having thestructure:

5′-X₁-Z-X₂-X₃-X₄-Z-X₅-3′  Formula 4

wherein X₁ and X₅ are independently 1-3 nucleotides wherein theinternucleotide linkages are optionally phosphorothioate; Z is anapthylene-azo compound; X₂ and X₄ are independently 1-5 nucleotideswherein the internucleotide linkages are optionally phosphorothioate;and X₃ is 10-25 nucleotides.

In one embodiment, a third modification can be inserted around themiddle of the antisense oligonucleotide. For longer nucleotides (greaterthan 25 bases), additional modifications could be used at intervals toconfer greater stability.

In the modifications of the invention, a modifying group is insertedbetween adjacent bases, thereby generating an ASO with reduced toxicityand improved affinity and stability. The bases can be DNA, 2′OMe RNA, orother modified bases. However, modified bases do not need to beemployed. Because the modifications are inserted between the bases, theycan be added as a phosphoramidite compound using standardphosphoramidite synthesis chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gel photograph that illustrates the levels of degradation ofsynthetic DNA oligomers in fetal bovine serum. A series of 10-mersingle-stranded DNA oligonucleotides were trace labeled with ³²P attheir 5′-ends and were incubated in serum at 37° C. for the indicatedtimes (0-240 minutes). Reaction products were separated bypolyacrylamide gel electrophoresis (PAGE) and visualized byphosphorimaging. Samples are identified in Table 4.

FIG. 2 illustrates relative miR-21 suppression by various anti-miRNAoligonucleotides (AMOs) using a luciferase reporter assay. A reporterplasmid that expresses both Renilla luciferase and firefly luciferasewas transfected into HeLa cells. Cell extracts were studied for relativeactivity of both enzymes and Renilla luciferase activity was normalizedto firefly luciferase activity. The Renilla luciferase gene contains amiR-21 binding site and miR-21 is highly expressed in HeLa cells.Different anti-miR-21 oligonucleotides (X-axis) were transfected intothe cells and the relative ability of different designs to suppressmiR-21 activity directly relate to the increase in Renilla luciferaseactivity (Y-axis).

FIG. 3 illustrates relative miR-21 suppression by various AMOs using aluciferase reporter assay comparing perfect match and compounds having1, 2, or 3 mismatches (mismatch pattern 1). A reporter plasmid thatexpresses both Renilla luciferase and firefly luciferase was transfectedinto HeLa cells. Cell extracts were studied for relative activity ofboth enzymes and Renilla luciferase activity was normalized to fireflyluciferase activity. The Renilla luciferase gene contains a miR-21binding site and miR-21 is highly expressed in HeLa cells. Differentanti-miR-21 oligonucleotides (X-axis) were transfected into the cells,and the relative ability of different designs to suppress miR-21activity directly relate to the increase in Renilla luciferase activity(Y-axis).

FIG. 4 illustrates relative miR-21 suppression by various AMOs using aluciferase reporter assay comparing perfect match and compounds having1, 2, or 3 mismatches (mismatch pattern 2). A reporter plasmid thatexpresses both Renilla luciferase and firefly luciferase was transfectedinto HeLa cells. Cell extracts were studied for relative activity ofboth enzymes, and Renilla luciferase activity was normalized to fireflyluciferase activity. The Renilla luciferase gene contains a miR-21binding site and miR-21 is highly expressed in HeLa cells. Differentanti-miR-21 oligonucleotides (X-axis) were transfected into the cells,and the relative ability of different designs to suppress miR-21activity directly relate to the increase in Renilla luciferase activity(Y-axis).

FIG. 5 illustrates knockdown of HPRT expression by DNA ASOs, with orwithout PS bonds or iFQ modification. ASOs were transfected into HeLacells and RNA was prepared 24 hours post transfection. Relative HPRTlevels were assessed by RT-qPCR and are reported on the Y-axis.

FIG. 6 illustrates knockdown of HPRT expression by chimeric “gapmer”ASOs, with or without PS bonds and with or without iFQ modification.ASOs were transfected into HeLa cells and RNA was prepared 24 hours posttransfection. Relative HPRT levels were assessed by RT-qPCR and arereported on the Y-axis.

FIG. 7 illustrates knockdown of HPRT using DsiRNAs at doses ranging from0.01 nM to 1.0 nM. DsiRNAs were modified with iFQ group(s) at positionswithin the duplexes as indicated in the schematic below the graph.DsiRNAs were transfected into HeLa cells and RNA was prepared 24 hourspost transfection. Relative HPRT levels were assessed by RT-qPCR and arereported on the Y-axis.

FIG. 8 illustrates the toxicity profiles of various AMO chemistries whentransfected for 24 hours at 50 nM or 100 nM in HeLa cells. The negativecontrol or “NC1” sequence is not predicted to target any known humanmiRNAs or mRNAs, and so toxicity effects should be specific to thechemical composition of the oligonucleotide. The MultiTox-Glo MultiplexCytotoxicity Assay was employed to measure cell viability followingtreatment with various chemically modified oligonucleotides (X-axis),and cell viability was calculated as a ratio of live/dead cells tonormalize the data independent of cell number (Y-axis). A decrease oflive/dead cell values correlates with decreased cell viability.

FIG. 9 illustrates apoptosis induction profiles caused by various AMOchemistries when transfected for 24 hours at 50 nM or 100 nM in HeLacells. The negative control or “NC1” sequence is not predicted to targetany known human miRNAs or mRNAs, and so induction of apoptosis should bespecific to the biological effects of chemical composition of theoligonucleotide in the cell. The Caspase-Glo 3/7 Assay was employed tomeasure the levels of caspase-3 and caspase-7, which are known effectorsof apoptosis, using a luciferase assay. Apoptosis induction followingtreatment with various chemically modified oligonucleotides (X-axis) isproportional to increasing levels of luminescence (Y-axis).

DETAILED DESCRIPTION OF THE INVENTION

The antisense oligonucleotides of the invention have modificationsplaced between nucleotides, wherein the modifications increase affinityto the complementary target and provide nuclease resistance. In oneembodiment of the invention, the compounds are the same as thosedescribed in U.S. application Ser. No. 13/073,866, the disclosure ofwhich is incorporated by reference herein in its entirety.

In another embodiment of the invention, the antisense oligonucleotidecomprises at least one modification that has the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁-R₅ areindependently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, an electron donatinggroup, or an attachment point for a ligand; and X is a nitrogen orcarbon atom, wherein if X is a carbon atom, the fourth substituentattached to the carbon atom can be hydrogen or a C1-C8 alkyl group. In afurther embodiment of the invention, the antisense oligonucleotidecomprises at least one modification that has the structure:

wherein the linking groups L₁ and L₂ positioning the modification at aninternal position of the oligonucleotide are independently an alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, or alkoxy groups; R₁, R₂, R₄,R₅ are independently a hydrogen, alkyl, alkynyl, alkenyl, heteroalkyl,substituted alkyl, aryl, heteroaryl, substituted aryl, cycloalkyl,alkylaryl, alkoxy, an electron withdrawing group, or an electrondonating group; R₆, R₇, R₉-R₁₂ are independently a hydrogen, alkyl,alkynyl, alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl,substituted aryl, cycloalkyl, alkylaryl, alkoxy, an electron withdrawinggroup, or an electron donating group; R₈ is a hydrogen, alkyl, alkynyl,alkenyl, heteroalkyl, substituted alkyl, aryl, heteroaryl, substitutedaryl, cycloalkyl, alkylaryl, alkoxy, or an electron withdrawing group;and X is a nitrogen or carbon atom, wherein if X is a carbon atom, thefourth substituent attached to the carbon atom can be hydrogen or aC1-C8 alkyl group.

The compositions and methods of the invention involve modification of anoligonucleotide by placing non-base modifying group(s) as insertionsbetween bases while retaining the ability of that sequence to hybridizeto a complementary sequence. Typically, insertion of non-base modifyinggroups between bases results in a significant loss of affinity of themodified sequence to its complement. The unique compositions of theinvention increase affinity of the modified sequence to its complement,increasing stability and increasing T_(m). Placement of such non-basemodifying group(s) between bases prevents nucleases from initiatingdegradation at the modified linkage(s). When placed between the firstand second bases at both ends of the oligonucleotide, the sequence isprotected from attack by both 5′-exonucleases and 3′-exonucleases.Placement at central position(s) within the sequence can additionallyconfer some resistance to endonucleases. In particular, compounds of theclass of Formula 2 above impede nuclease attack for several flankinginternucleotide phosphate bonds adjacent to the modified linkage,creating a protected “zone” where unmodified linkages are lesssusceptible to nuclease cleavage. The modifications also preventnucleases from cleaving terminal bases. Thus the compositions andmethods of the invention permit synthesis of ASOs having increased T_(m)and increased nuclease resistance yet do not employ modified but insteademploy a non-base modifying group inserted between residues.

The ability of the modifying groups of the present invention to increasebinding affinity (T_(m)) of duplexed nucleic acids is demonstrated inExample 1, where melting studies were conducted for a series ofunmodified and modified 10-mer duplex DNA oligomers. Using compositionsand methods of the present invention, an increase of +11° C. wasachieved using only two modifying groups (between the two terminal baseson each end of the oligomer). Similar duplexes made with insertions of apropanediol group show significant destabilization, consistent with theexpected results for non-base insertions.

The ability of the modifying groups of the present invention to improvenuclease stability is demonstrated in Example 2, where single-strandedDNA oligomers were incubated in serum (subjected to degradation by serumnucleases) and then examined for integrity by polyacrylamide gelelectrophoresis (PAGE). Unmodified DNA oligomers are rapidly degraded inserum whereas a 10-mer DNA oligonucleotide with an insertion of thenapthylene-azo modifier between the terminal bases on each end resultedin a compound that was not degraded after 4 hours incubation. Othermodifying groups, such as a propanediol spacer, only slowed the rate ofdegradation slightly. T_(m)-enhancing, nuclease blocking modifications(such as the napthylene-azo group) can be inserted into single-strandedoligomers to improve properties. Stabilized, increased binding affinityoligomers of this type can have a variety of uses, as is wellappreciated by those with skill in the art. As examples (not meant to belimiting), such oligomers can be used as ASOs to promote reduction ofmRNA or miRNA levels in a cell or animal. Such examples are demonstratedin Examples 3 and 4 below.

In a further embodiment of the invention, the modifications could alsobe incorporated into double-stranded nucleic acids, such as syntheticsiRNAs and miRNAs. Careful placement of the modifying group should leadto improvements in nuclease stability and could alter local thermalstability, which if employed asymmetrically in an RNA duplex, is wellknown to influence strand loading into RISC (Peek and Behlke, 2007,Curr. Opin. Mol. Ther. 9(2): 110-18), and therefore impact relativebiological potency of the compound as a synthetic trigger of RNAi.

Oligonucleotides antisense in orientation to miRNAs will bind the miRNAand functionally remove that species from participation in themicroRNA-Induced Silencing Complex (miRISC) (Krutzfeldt et al., 2007,Nucleic Acids Res. 35(9): 2885-92). Such anti-miRNA oligonucleotides(AMOs) are thought to function by a steric binding mechanism, andcompounds with high stability and high affinity generally show improvedfunctional performance compared with low affinity compounds (Lennox andBehlke, 2010, Pharm. Res. 27(9): 1788-99). The ASOs of the presentinvention can function as anti-miRNA oligonucleotides.

In the modifications of the present invention, a modifying group isinserted between adjacent bases, thereby generating an ASO with reducedtoxicity and improved binding affinity and nuclease stability. The basescan be DNA, 2′OMe RNA, LNA, or other modified bases. However, modifiedbases do not need to be employed. Because the modifications are insertedbetween the bases, they can be added as a phosphoramidite compound usingstandard phosphoramidite synthesis chemistry.

In yet another application where ASOs are employed to alter or modifygene expression, the ASOs are designed to be complementary to a pre-mRNAspecies at sites at or near an intron/exon splice junction. Binding ofthe ASO at or near splice sites can alter processing at this intron/exonjunction by the nuclear splicing machinery thereby changing splicepatterns present in the final mature mRNA (i.e., can be used to alterthe exons that are included or excluded in the final processed mRNA).Following mRNA maturation, the altered mRNA will direct synthesis of analtered protein species as a result of this ASO treatment. Methods todesign splice-blocking oligonucleotides (SBOs) are well known to thosewith skill in the art (see, e.g., Aartsma-Rus et al., 2009, Mol. Ther.17(3): 548-53; and Mitrpant et al., 2009, Mol. Ther. 17(8): 1418-26).Because SBOs are intended to alter the form of an mRNA but not destroythat mRNA, oligonucleotides of this class are made using chemistrieswhich are compatible with steric blocking antisense mechanism of actionand not chemistries or designs that trigger RNA degradation. One exampleof the use of SBOs induces exon-skipping in the dystrophin gene inindividuals having a mutant form of this gene which causes Duchene'sMuscular Dystrophy (see Muntoni and Wood, 2011, Nat. Rev. Drug Discov.10(8): 621-37; and Goemans et al., 2011, N. Engl. J. Med. 364(16):1513-22). Synthetic oligonucleotides using the design and chemistries ofthe present invention can be employed as SBOs.

In one embodiment, a synthetic oligonucleotide comprises anon-nucleotide modifier of the present invention positioned at or nearone or both ends of the sequence. In another embodiment, a syntheticoligonucleotide comprises a non-nucleotide modifier of the presentinvention positioned between a terminal base and a penultimate base ofeither the 3′- or the 5′-end of the oligonucleotide. In a furtherembodiment, the oligonucleotide contains a modification between theterminal base and the penultimate base of both the 3′- and 5′-ends.

In one embodiment of the invention, the modification is a napthylene-azocompound. The oligonucleotide is made using modified bases such that thecomplex of the SBO with the target pre-mRNA does not form a substratefor RNase H, using chemically-modified residues that are well known tothose with skill in the art, including, for example, 2′-O-methyl RNA,2′-methyoxyethyl RNA (2′-MOE), 2′-F RNA, LNA, and the like. SBOs madeusing the non-nucleotide modifiers of the present invention haveincreased binding affinity compared to the cognate unmodified species.This can permit use of shorter sequences, which can show improved uptakeinto cells and improved biological activity.

In another embodiment of the invention, the modification has thestructure:

In a further embodiment of the invention, the modification has thestructure:

The antisense oligonucleotides of the invention may be conjugated toother ligands, which may aid in the delivery of the antisenseoligonucleotide to a cell or organism. In one embodiment of theinvention, the ligand is 5′ cholesterol monoethyleneglycol (/5CholMEG/):

In another embodiment of the invention, the ligand is 5′ cholesteroltriethyleneglycol (/5Chol-TEG/):

In a further embodiment of the invention, the ligand is 3′ cholesterolmonoethyleneglycol (/3CholMEG/):

In another embodiment of the invention, the ligand is 3′ cholesteroltriethyleneglycol (/3CholTEG/):

The ligand may be conjugated to the antisense oligonucleotide with orwithout an additional S18 (hexaethyleneglycol) spacer. In a preferredembodiment, the antisense oligonucleotide is an anti-miRNAoligonucleotide (AMO). In another preferred embodiment, thenon-nucleotide modification is a FQ napthylene-azo compound (alsoreferred to as iFQ or ZEN in this disclosure).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the improved thermal stability of internalnapthylene-azo-containing oligomers compared to other compounds.

Oligonucleotide synthesis and preparation. DNA oligonucleotides weresynthesized using solid phase phosphoramidite chemistry, deprotected anddesalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.)according to routine techniques (Caruthers et al., 1992, MethodsEnzymol. 211: 3-20). The oligomers were purified using reversed-phasehigh performance liquid chromatography (RP-HPLC). The purity of eacholigomer was determined by capillary electrophoresis (CE) carried out ona Beckman P/ACE MDQ system (Beckman Coulter, Inc., Fullerton, Calif.).All single-strand oligomers were at least 90% pure.Electrospray-ionization liquid chromatography mass spectrometry(ESI-LCMS) of the oligonucleotides was conducted using an Oligo HTCSsystem (Novatia, Princeton, N.J.), which consisted of ThermoFinniganTSQ7000, Xcalibur data system, ProMass data processing software, andParadigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.). Protocolsrecommended by the manufacturers were followed. Experimental molarmasses for all single-strand oligomers were within 1.5 g/mol of expectedmolar mass. These results confirm identity of the oligomers.

Preparation of DNA samples. Melting experiments were carried out inbuffer containing 3.87 mM NaH₂PO₄, 6.13 mM Na₂HPO₄, 1 mM Na₂EDTA, and1000 mM NaCl. 1 M NaOH was used to titrate each solution to pH 7.0.Total sodium concentrations were estimated to be 1020 mM. The DNAsamples were thoroughly dialyzed against melting buffer in a 28-wellMicrodialysis System (Life Technologies, Carlsbad, Calif.) following themanufacturer's recommended protocol. Concentrations of DNA oligomerswere estimated from the samples' UV absorbance at 260 nm in aspectrophotometer (Beckman Coulter, Inc., Fullerton, Calif.), usingextinction coefficients for each oligonucleotide that were estimatedusing the nearest neighbor model for calculating extinction coefficients(see Warshaw et al., 1966, J. Mol. Biol. 20(1): 29-38).

Internal modifications studied. The FQ napthylene-azo compound (Formula3, Integrated DNA Technologies, Inc., sometimes referred to as “iFQ” or“ZEN” in this disclosure), was introduced into oligonucleotides usingphosphoramidite reagents at the time of synthesis.

In the first series of duplexes, the iFQ group was placed as aninsertion between bases in the duplex so that a 10-base top strandannealed to a 10-base bottom strand and the iFQ group was not aligned toa base. Additionally, 10-mer oligonucleotides with C3 spacer insertionswere also synthesized and studied. The C3 spacer represents the controlwherein a linear insertion of a phosphate group plus propanediol isplaced between bases, which is similar to the iFQ insertions withouthaving the napthylene-azo ring structures present. Extinctioncoefficients at 260 nm of iFQ were estimated to be 13340; the C3 spacerdoes not contribute to UV absorbance.

In a second series of duplexes, the iFQ group was placed as asubstitution for a base in the duplex so that a 9-base top strandannealed to a 10-base bottom strand and the iFQ group was aligned to abase. Additionally, 10-mer oligonucleotides with C3 spacer substitutionswere also synthesized and studied.

Measurement of melting curves. Oligomer concentrations were measured atleast twice for each sample. If the estimated concentrations for anysample differed more than 4%, the results were discarded and newabsorbance measurements were performed. To prepare oligonucleotideduplexes, complementary DNA oligomers were mixed in 1:1 molar ratio,heated to 367 K (i.e., 94° C.) and slowly cooled to an ambienttemperature. Each solution of duplex DNA was diluted with melting bufferto a total DNA concentration (C_(T)) of 2 μM.

Melting experiments were conducted on a single beam Beckman DU 650spectrophotometer (Beckman-Coulter) with a Micro T_(m) Analysisaccessory, a Beckman High Performance Peltier Controller (to regulatethe temperature), and 1 cm path-length cuvettes. Melt data were recordedusing a PC interfaced to the spectrophotometer. UV-absorbance values at268 nm wavelength were measured at 0.1 degree increments in thetemperature range from 383 to 368 K (i.e., 10-95° C.). Both heating(i.e., “denaturation”) and cooling (i.e., “renaturation”) transitioncurves were recorded in each sample at a controlled rate of temperaturechange (24.9±0.3° C. per hour). Sample temperatures were collected fromthe internal probe located inside the Peltier holder, and recorded witheach sample's UV-absorbance data. Melting profiles were also recordedfor samples of buffer alone (no oligonucleotide), and these “blank”profiles were digitally subtracted from melting curves of the DNAsamples. To minimize systematic errors, at least two melting curves werecollected for each sample in different cuvettes and in differentpositions within the Peltier holder.

Determination of melting temperatures. To determine each sample'smelting temperature, the melting profiles were analyzed using methodsthat have been previously described (see Doktycz et al., 1992,Biopolymers 32(7): 849-64; Owczarzy et al., 1997, Biopolymers 44(3):217-39; and Owczarzy, 2005, Biophys. Chem. 117(3): 207-15.). Briefly,the experimental data for each sample was smoothed, using a digitalfilter, to obtain a plot of the sample's UV-absorbance as a function ofits temperature. The fraction of single-stranded oligonucleotidemolecules, θ, was then calculated from that plot. The “meltingtemperature” or “T_(m)” of a sample was defined as the temperature whereθ=0.5. Table 1 lists the melting temperatures of the oligonucleotidestested.

TABLE 1  Melting temperatures for nucleic acids containinga single internal modifying group as an insertion SEQ Avg ID NO:Sequence T_(m) ΔT_(m) AT_(m) 1 5′ ATCGTTGCTA 43.9 — 2 3′ TAGCAACGAT 3 5′ATC/GTTGCTA iFQ “/” 48.0 4.1 +3.7 2 3′ TAG CAACGAT 4 5′ATCG/TTGCTA iFQ “/” 48.6 4.7 2 3′ TAGC AACGAT 5 5′ ATCGT/TGCTA iFQ “/”46.3 2.4 2 3′ TAGCA ACGAT 6 5′ A/TCGTTGCTA iFQ “/” 51.75 7.9 +7.2 2 3′T AGCAACGAT 7 5′ ATCGTTGCT/A iFQ “/” 50.25 6.4 2 3′ TAGCAACGA T 8 5′ATC/GTTGCTA iSpC3 “/” 36.3 −7.6 −8.7 2 3′ TAG CAACGAT 9 5′ATCG/TTGCTA iSpC3 “/” 36.6 −7.3 2 3′ TAGC AACGAT 10 5′ATCGT/TGCTA iSpC3 “/” 32.6 −11.3 2 3′ TAGCA ACGAT “/” signifies the siteof insertion of a modifying group between bases as indicated. iFQ =internal FQ azo quencher (ZEN) iSpC3 = internal C3 spacer

When the iFQ (ZEN) modifier was inserted centrally within a 10-meroligonucleotide (between bases 3/4, 4/5, or 5/6), T_(m) was increased byan average of 3.7° C. When placed between terminal residues (betweenbases 1/2 or 9/10), T_(m) was increased by an average of 7.2° C. Incontrast, insertion of a small propanediol group (C3 spacer) had asignificant negative impact on the T_(m) of the duplex (average ΔT_(m)of −8.7° C.).

A subset of these sequences were studied using the internalmodifications as base substitutions, such that now a 9-base top strandannealed to a 10-base bottom strand with the modification replacing abase and being aligned with a base on the opposing strand. Results areshown in Table 2. In this case, it is evident that the base substitutionwas significantly destabilizing whereas the insertions (Table 1) werestabilizing (ZEN) or were at least less destabilizing (C3).

TABLE 2  Melting temperatures for nucleic acids containinga single internal modifying group comparing substitution vs. insertionSEQ Ins ID vs. NO: Duplex Sequence Subs T_(m) ΔT_(m) 1 5′-ATCGTTGCTA-3′— 43.9 0.0 2 3′-TAGCAACGAT-5′ 3 5′-ATC/GTTGCTA-3′ iFQ “/” I 48.0 4.1 23′-TAG CAACGAT-5′ 8 5′-ATC/GTTGCTA-3′ iSpC3 “/” I 36.3 −7.6 23′-TAG CAACGAT-5′ 11 5′-ATC/TTGCTA-3′ iFQ “/” S 34.7 −9.2 23′-TAGCAACGAT-5′ 12 5′-ATC/TTGCTA-3′ iSpC3 “/” S <20 2 3′-TAGCAACGAT-5′13 5′-ATCG/TTGCTA-3′ iFQ “/” I 48.6 4.7 2 3′-TAGC AACGAT-5′ 145′-ATCG/TTGCTA-3′ iSpC3 “/” I 36.6 −7.3 2 3′-TAGC AACGAT-5′ 155′-ATCG/TGCTA-3′ iFQ “/” S 38.2 −5.7 2 3′-TAGCAACGAT-5′ 165′-ATCG/TGCTA-3′ iSpC3 “/” S <24 2 3′-TAGCAACGAT-5′ 17 5′-ATCGT/TGCTA-3′iFQ “/” I 46.3 2.4 2 3′-TAGCA ACGAT-5′ 18 5′-ATCGT/TGCTA-3′ iSpC3 “/” I32.6 −11.3 2 3′-TAGCA ACGAT-5′ 19 5′-ATCGT/GCTA-3′ iFQ “/” S 40.8 −3.1 23′-TAGCAACGAT-5′ 20 5′-ATCGT/GCTA-3′ iSpC3 “/” S <26 2 3′-TAGCAACGAT-5′

For this series of internal modifications, the average ΔT_(m) for iFQ(ZEN) insertion was +3.7° C. while the average ΔT_(m) for iFQ (ZEN)substitution was −6° C. The average ΔT_(m) for iC3 spacer insertion was−8.7° C. while the average ΔT_(m) for iC3 spacer substitution was morethan −20° C. (accurate measurements were not possible as the T_(m) wasbelow room temperature). Therefore insertion placement is preferred tosubstitution placement.

The napthylene-azo modifier was introduced into the same 10-mer oligomersequence at 2 or 3 sites, either adjacent to or separated by severalbases. Duplexes were formed and T_(m) values were measured as before.Results are shown in Table 3. Some of the singly modified duplexes fromTable 1 are also included in Table 3 to improve clarity of comparisonsbetween modification patterns.

TABLE 3  Melting temperatures for nucleicacids containing multiple internal modifying groups as insertions SEQ IDNO: Sequence Tm ΔTm 1 5′ ATCGTTGCTA 43.87 — 2 3′ TAGCAACGAT 3 5′ATC/GTTGCTA 1x iFQ “/” 48.02 4.15 2 3′ TAG CAACGAT 21 5′ATC//GTTGCTA 2x iFQ “//” 39.62 −4.25 2 3′ TAG  CAACGAT 22 5′ATC/GTT/GCTA 2x iFQ “/.../” 46.72 2.8 2 3′ TAG CAA CGAT 23 5′ATC/GT/TG/CTA 3x iFQ “/../../” 43.36 −0.51 2 3′ TAG CA AC GAT 13 5′ATCG/TTGCTA 1x iFQ “/” 48.57 4.70 2 3′ TAGC AACGAT 24 5′ATCG//TTGCTA 2x iFQ “//” 39.82 −4.05 2 3′ TAGC  AACGAT 17 5′ATCGT/TGCTA 1x iFQ “/” 46.32 2.45 2 3′ TAGCA ACGAT 25 5′ATCGT//TGCTA 2x iFQ “/” 36.76 −8.90 2 3′ TAGCA  ACGAT 26 5′A/TCGTTGCTA 1x iFQ “/” 51.75 7.88 2 3′ T AGCAACGAT 27 5′ATCGTTGCT/A 1x iFQ “/” 50.25 6.38 2 3′ TAGCAACGA T 28 5′A/TCGTTGCT/A 2x iFQ “/” 54.91 11.04 2 3′ T AGCAACGA T “/” signifies thesite of insertion of a modifying group between bases as indicated.

Insertion of two adjacent napthylene-azo modifiers was destabilizing andT_(m) was found to change by −4 to −8.9° C. depending on sequencecontext. Placing two napthylene-azo modifying groups in the samesequence separated by 3 bases was slightly stabilizing (T_(m)+2.9° C.);however, this was less stabilizing than use of a single modifier alone(T_(m)+4.7° C.). Use of 3 modifier groups separated by 2 bases betweengroups was destabilizing. However, when two napthylene-azo modifiergroups were placed at the ends (between both bases 1/2 and 9/10), T_(m)was increased by 11° C. Thus, an additive effect can be obtained byplacing multiple insertions of the modifying group into a sequence solong as a sufficient number of bases separate the groups. End effectsare particularly potent.

Therefore, internal incorporation of the napthylene-azo group within aDNA duplex stabilizes the duplex when placed as an insertion betweenbases. Certain anthraquinone groups can stabilize a duplex when placedon the ends (Patra et al., 2009, J. Am. Chem. Soc. 131(35): 12671-81);however, this effect has not been described for internal placement.Therefore, the use of napthylene-azo-class compounds would be preferredas an internal modifying group to increase duplex stability.

EXAMPLE 2

This example demonstrates the improved nuclease stability of internalnapthylene-azo-containing oligomers compared to other compounds.

Oligonucleotide synthesis and purification. DNA oligonucleotides weresynthesized using solid phase phosphoramidite chemistry, deprotected anddesalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.)according to routine techniques (Caruthers et al., 1992). The oligomerswere purified using reversed-phase high performance liquidchromatography (RP-HPLC). The purity of each oligomer was determined bycapillary electrophoresis (CE) carried out on a Beckman P/ACE MDQ system(Beckman Coulter, Inc., Fullerton, Calif.). All single-strand oligomerswere at least 90% pure. Electrospray-ionization liquid chromatographymass spectrometry (ESI-LCMS) of the oligonucleotides was conducted usingan Oligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.The synthesized oligonucleotides are listed in Table 4.

TABLE 4  Synthetic oligomers employed in Example 2 SEQ ID NO: NameSequence 1 DNA 5′ ATCGTTGCTA 3′ 26 5′ iFQ 5′ A(iFQ)TCGTTGCTA 3′ 27 3′iFQ 5′ ATCGTTGCT(iFQ)A 3′ 28 5′ + 3′ iFQ 5′ A(iFQ)TCGTTGCT(iFQ)A 3′ 295′ iC3 5′ A(iSpC3)TCGTTGCTA 3′ 30 3′ iC3 5′ ATCGTTGCT(iSpC3)A 3′ 31 5′ +3′ iC3 5′ A(iSpC3)TCGTTGCT(iSpC3)A 3′

Radiolabeling of oligomers. Single-stranded oligomers were radiolabeledat the 5′-end using polynucleotide kinase. Briefly, 5 pmoles of eacholigonucleotide were incubated with 10 units of OptiKinase (USB,Cleveland, Ohio) and 10 μmoles of alpha ³²P γ-ATP (3000 Ci/mmol) (PerkinElmer, Waltham, Mass.) for 30 minutes at 37° C., followed by 65° C. for10 minutes. Excess radionucleotide was removed by gel filtration usingtwo sequential passes through MicroSpin G-25 columns (GE Healthcare,Buckinghamshire, UK). Isotope incorporation was measured in a PerkinElmer TriCarb 2800 TR scintillation counter (Perkin Elmer, Waltham,Mass.).

Serum degradation of oligomers. As labeling efficiencies varied (lowerspecific activity was obtained for the oligomers with a modificationnear the 5′-end), equivalent numbers of dpms of radiolabeled oligomerswere mixed with unlabeled oligomers to a final concentration of 8 μM inthe presence of 50% fetal bovine serum (not heat inactivated;Invitrogen, Carlsbad, Calif.). Samples were incubated at 37° C. for 0,30, 60, or 240 minutes; aliquots were removed at the indicated timepoints, an equal volume of 90% formamide was added, and samples flashfrozen on dry ice. Degradation products were separated by PAGE using a20% polyacrylamide, 7 M Urea denaturing gel and visualized on a Cyclonephosphorimager (Perkin Elmer, Waltham, Mass.). Results are shown in FIG.1.

The unmodified DNA oligomer was rapidly degraded and no intactfull-length material was present after 30 minutes incubation. The samplewas fully degraded by 4 hours. A similar pattern of degradation was seenfor the oligomer having a single internal C3 spacer positioned near the5′-end. In contrast, only incomplete degradation was observed for theoligomer bearing a single internal FQ modifier near the 5′-end. Thedegradation pattern observed is most consistent with processive3′-exonuclease cleavage that stopped before the oligomer was fullydegraded. This suggests the possibility that the iFQ modifier protectsneighboring DNA residues from exonuclease degradation, providing a smallzone of protection around the 5′-end.

The oligomer having a single internal C3 spacer near the 3′-end showsprompt removal of what appears to be a single base and then was slowlydegraded. Slightly greater protection was seen in the oligomer having aninternal C3 spacer placed near both ends. In contrast, no evidence wasseen for single base cleavage at the 3′-end of the oligomer having asingle internal FQ modifier near the 3′-end, and no evidence fordegradation was observed after 4 hours incubation in 50% serum for theoligomer having an internal FQ modifier placed near both ends.

Therefore, the FQ modifier will block exonuclease attack from theenzymes present in fetal bovine serum, and can confer relative nucleaseresistance to neighboring unmodified bases, creating a protected “zone”in its vicinity.

EXAMPLE 3

This example demonstrates improved functional activity of internalnapthylene-azo-containing ASOs at reducing microRNA activity compared toother compounds.

Oligonucleotide synthesis and purification. DNA, 2′OMe RNA, and LNAcontaining oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted on NAP-5 columns(Amersham Pharmacia Biotech, Piscataway, N.J.) according to routinetechniques (Caruthers et al., 1992). The oligomers were purified usingreversed-phase high performance liquid chromatography (RP-HPLC). Thepurity of each oligomer was determined by capillary electrophoresis (CE)carried out on a Beckman P/ACE MDQ system (Beckman Coulter, Inc.,Fullerton, Calif.). All single-strand oligomers were at least 85% pure.Electrospray-ionization liquid chromatography mass spectrometry(ESI-LCMS) of the oligonucleotides was conducted using an Oligo HTCSsystem (Novatia, Princeton, N.J.), which consisted of ThermoFinniganTSQ7000, Xcalibur data system, ProMass data processing software, andParadigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.). Protocolsrecommended by the manufacturers were followed. Experimental molarmasses for all single-strand oligomers were within 1.5 g/mol of expectedmolar mass. These results confirm identity of the oligomers. Table 5lists the synthetic oligomers used in this Example.

TABLE 5  Synthetic oligomers employed in Example 3 (miR21 AMOs) SEQ IDNO: Name Sequence 32 2′OMe U C A A C A U C A G U C U G A U A A G C U A33 2′OMe PSends U*C*A*A C A U C A G U C U G A U A A G*C*U*A 34 2′OMe PSU*C*A*A*C*A*U*C*A*G*U*C*U*G*A*U*A*A*G*C*U*A 35 2′OMe 5′ + 3′iFQU_(z)C A A C A U C A G U C U G A U A A G C U_(z)A 36 2′OMePS 5′ + 3′iFQU_(z)C*A*A*C*A*U*C*A*G*U*C*U*G*A*U*A*A*G*C*U_(z)A 37 2′OMe 3′iFQU C A A C A U C A G U C U G A U A A G C U_(z)A 38 2′OMe 5′iFQU_(z)C A A C A U C A G U C U G A U A A G C U A 39 2′OMe 5′ + I + 3′iFQU_(z)C A A C A U C A G U_(z)C U G A U A A G C U_(z)A 40 DNA/LNA PSt*C*a*a*C*a*t*C*a*g*T*c*t*G*a*t*A*a*g*C*t*a 41 2′OMe/LNA PSU*C*A*A*C*A*U*C*A*G*T*C*U*G*A*U*A*A*G*C*U*A Uppercase = 2′OMe RNALowercase = DNA Uppercase with underscore = LNA “*” = phosphorothioatelinkage “z” = napthylene-azo modifier (iFQ)

Plasmid preparation. The psiCHECK™-2 vector (Promega, Madison, Wis.) wasrestriction enzyme digested sequentially with Xhol and Notl (New EnglandBiolabs, Ipswitch, Mass.) and purified with a Qiaquick PCR purificationcolumn (Qiagen, Valencia, Calif.). A perfect complement hsa-miR-21binding site was created by annealing two synthetic duplexedoligonucleotides (Integrated DNA Technologies, Coralville, Iowa) and wascloned into the Xhol/Notl sites in the 3′UTR of Renilla luciferase. ThismiR-21 reporter construct was sequence verified on a 3130 GeneticAnalyzer (AB, Foster City, Calif.). Plasmids were purified using aPlasmid Midiprep Kit (Bio-Rad, Hercules, Calif.) and treated twice forendotoxin removal with the MiraCLEAN Endotoxin Removal Kit (MinisCorporation, Madison, Wis.). Plasmids were filtered through a 0.2 μfilter and quantified by measurement of the absorbance at 260 nm usingUV spectrophotometry. This reporter plasmid having a perfect match miRNAbinding site is denoted as psiCHECK™-2-miR21.

Cell culture, transfections, and luciferase assays. HeLa cells wereplated in a 100 mm dish in DMEM containing 10% FBS to achieve 90%confluency the next day. The following morning, 5 μg of thepsiCHECKT™-2-miR21 plasmid was transfected with Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.). After 6 hours, cells were washed with 1×PBS, trypsinized, counted, and replated in DMEM with 10% FBS in 48-wellplates to achieve ˜70% confluency the next day. The following morning,the miR-21 AMOs were transfected at various concentrations in triplicatewith 1 μl TriFECTin® (Integrated DNA Technologies) per well in DMEMwithout serum. After 6 hours, the transfection media was removed andreplenished with DMEM containing 10% FBS. The following morning, (48hours after plasmid transfection, 24 hours after miRNA AMO transfection)the cells were analyzed for luciferase luminescence using theDual-Luciferase® Reporter Assay System (Promega, Madison, Wis.) per themanufacturer's instructions. Renilla luciferase was measured as a foldincrease in expression compared to the TriFECTin® reagent-only negativecontrols. Values for Renilla luciferase luminescence were normalized tolevels concurrently measured for firefly luciferase, which is present asa separate expression unit on the same plasmids as an internal control(RLuc/FLuc ratio).

Results. The RLuc/FLuc ratios obtained from transfections done with thevarious AMOs are shown in FIG. 2. In the untreated state, HeLa cellscontain large amounts of miRNA 21 that suppress expression of the RLucreporter. Any treatment that decreases miR-21 levels leads to anincrease in RLuc expression and thus increases the relative RLuc/FLucratio (with FLuc serving as an internal normalization control fortransfection efficiency).

The unmodified 2′OMe RNA AMO showed essentially no inhibition of miR-21activity, probably due to rapid nuclease degradation of this unprotectedoligomer during transfection or in the intracellular environment. Theaddition of 3 PS linkages on each end of the AMO blocks exonucleaseattack and the “2′OMe-PSends” AMO showed good potency for functionalknockdown of miR-21. When this AMO is changed to be fully PS modified(“2′OMe-PS”), potency drops, which is probably due to having lowerbinding affinity (lower T_(m)) that accompanies extensive PSmodification. Each substitution of a PS bond for a standardphosphodiester bond reduces T_(m), and there are 21 PS bonds in thisoligomer compared with only 6 PS bonds in the “2′OMe PSends” version.

A desirable modification chemistry or modification pattern is one thatboth increases nuclease stability and increases T_(m). The internalnapthylene-azo modifier meets these criteria. The 2′OMe oligomer havingan internal napthylene-azo modifier placed between the terminal andadjacent bases on each end (2′OMe 5′+3′iFQ) showed markedly improvedanti-miR21 activity and was more potent than any of the PS modified2′OMe AMOs tested. Adding PS modification to this design (2′OMePS5′+3′iFQ) reduced potency, likely due to the lower binding affinitycaused by the addition of 19 PS linkages. This compound was neverthelessstill significantly more potent than the 2′OMe-PS version without the 2iFQ modifications.

Protecting only one end of the anti-miR-21 AMO with an internalnapthylene-azo modifier showed improved potency when compared with theunmodified 2′OMe AMO; however, the performance was much reduced comparedwith the dual-modified version. Interestingly, modification at the5′-terminal linkage had more effect than modification at the 3′-terminallinkage, the exact opposite of the results anticipated from the relativeserum stability profiles demonstrated in Example 2. This result isexplained by measured effects of T_(m) (see Table 6).

Addition of a third iFQ modification into the end-blocked version (2′OMe5′+I+3′iFQ) showed reduced potency compared with the originalend-blocked version (2′OMe 5′+3′iFQ), which is likely due to a reductionof T_(m) seen with placing this many iFQ modifying groups in a single,short 22-mer sequence.

The “DNA/LNA-PS” AMO is a design employed by Exiqon as its preferredanti-miRNA agent and is widely accepted as the “gold standard” for miRNAknockdown studies performed today. The DNA/LNA compound showed the samepotency as the dual-modified “2′OMe 5′+3′iFQ” AMO. The “2′OMe/LNA-PS”AMO showed highest potency within the set studied. The LNA modificationconfers nuclease resistance and gives very large increases in T_(m),resulting in AMOs with higher potency but also having lower specificitythan AMOs without LNA bases with lower binding affinity. The relativespecificity of the different AMOsis presented in Example 4 below. Ofnote, the LNA-PS modified AMOs show some toxicity and cell culturestransfected with the highest doses (50 nM) had dysmorphic, unhealthyappearing cells at the time of harvest. The “2′OMe 5′+3′iFQ” AMO did notshow any visual evidence for toxicity at any of the doses tested. Insubsequent experimentation, toxicity effects were evaluated at highdoses by measuring cell viability, cytotoxicity, and induction ofapoptosis (see Example 7). The “2′OMe 5′+3′iFQ” chemistry showed nocellular toxicity, compared to the substantial cellular toxicity thatoccurred upon transfection of single-stranded oligonucleotidescontaining LNA bases, extensive PS modification (all 21 linkages), orboth LNA and PS modifications (the “gold standard” AMO). Thus, the“2′OMe 5′+3′iFQ” may be a new class of AMO that achieves high potencyyet maintains low toxicity.

The melting temperatures, T_(m), of the AMOs described above weremeasured using the same methods described in Example 1. Synthetic AMOoligonucleotides were annealed to a synthetic RNA complement (maturemiR21 sequence). Measurements were done at 2 μM duplex concentration in150 mM NaCl to approximate intracellular ion concentration.

TABLE 6  T_(m) of synthetic miR21 AMOs in 150 mN NaCl SEQ ID NO: NameSequence T_(m) ΔT_(m) 32 2′OMe U C A A C A U C A G U 72.1 —C U G A U A A G C U A 33 2′OMe U*C*A*A C A U C A G U 70.9 −1.2 PSendsC U G A U A A G*C*U*A 34 2′OMe PS U*C*A*A*C*A*U*C*A*G*U* 67.1 −5.0C*U*G*A*U*A*A*G*C*U*A 35 2′OMe U_(z)C A A C A U C A G U  75.4 +3.3 5′ +3′iFQ C U G A U A A G C U_(z)A 36 2′OMePS U_(z)C*A*A*C*A*U*C*A*G*U* 70.6-1.5 5′ + 3′iFQ C*U*G*A*U*A*A*G*C*U_(z)A 37 2′OMe 3′iFQU C A A C A U C A G U  72.4 +0.3 C U G A U A A G C U_(z)A 38 2′OMe 5′iFQU_(z)C A A C A U C A G U  74.3 +2.2 C U G A U A A G C U A 39 2′OMeU_(z)C A A C A U C A G U_(z)C 71.3 −0.8 5′ + I + U G A U A A G C U_(z)A3′iFQ 40 DNA/LNA t*C*a*a*C*a*t*C*a*g*T* 74.0 +1.9 PSc*t*G*a*t*A*a*g*C*t*a 41 2′OMe/LNA U*C*A*A*C*A*U*C*A*G*T* 85.9 +13.8 PSC*U*G*A*U*A*A*G*C*U*A Uppercase = 2′OMe RNA Lowercase = DNA Uppercasewith underscore = LNA “*” = phosphorothioate linkage “z” =napthylene-azo modifier (iFQ)

The 22-mer 2′OMe miR21 AMO showed a T_(m) of 72.1° C. when hybridized toan RNA perfect complement in 150 mM NaCl. Substitution of 6 PS bonds fornative PO linkages lowered T_(m) by 1.2° C. (“2′OMe PSends”) andcomplete PS modified lowered T_(m) by 5.0° C. (“2′OMe PS”), a change of−0.20 to −0.25° C. per modified internucleotide linkage. In contrast,insertion of an iFQ group at the 3′-terminal linkage (“2′OMe 3′iFQ”)resulted in a T_(m) increase of +0.3° C. and at the 5′-terminal linkage(“2′OMe 5′iFQ”) resulted in a T_(m) increase of +2.2° C. Combining thesetwo designs, addition of two iFQ modifications (one at each terminallinkage, “2′OMe 5′+3′iFQ”) increased T_(m) to 75.4° C., which is achange of +3.3° C. compared with the unmodified sequence or +4.5° C.relative to the PS-end blocked sequence (which is the most relevantcomparison). This dual-end-modification pattern results in good nucleaseresistance (FIG. 1) and when employed in a 2′OMe AMO shows increasedT_(m) (Table 6) and is a very potent anti-miR21 agent (FIG. 2).Interestingly, addition of a third iFQ group centrally placed (“2′OMe5′+I+3′ iFQ”) resulted in a T_(m) decrease of 0.8° C. relative to theunmodified compound, or a decrease of 4.1° C. relative to the two-endinsertion version (“2′OMe 5′+3′iFQ”). Thus while inserting the iFQmodifier between terminal bases increases T_(m) adding a thirdmodification in the center of the sequence leads to a decrease in T_(m)even though these modifications are fully 10 bases distant from eachother. This loss of T_(m) results in a loss of functional potency (FIG.2). Therefore the dual-modified end-insertion pattern is preferred.

As a general rule, the relative potency of the various miR21 AMOscorrelated with increased binding affinity (T_(m)). All variations inpotency observed between compounds could be explained by relativecontributions of improvements in binding affinity and nuclease stabilitybetween the different modification patterns studied. The AMO having2′OMe bases with an iFQ modification placed near each end (“2′OMe5′+3′iFQ”) provided an excellent balance of nuclease stability withincreased T_(m) and the only AMO showing higher potency was the“2′OMe/LNA-PS” compound. The “2′OMe/LNA-PS” compound, however, showedreduced specificity due to its extreme elevation in binding affinity(see Example 4) and increased cellular toxicity (see Example 7).Therefore, the novel “2′OMe 5′+3′iFQ” design of the present invention issuperior.

EXAMPLE 4

This example demonstrates improved specificity of internalnapthylene-azo-containing oligomers when reducing microRNA activitycompared to other compounds containing modifications that increasebinding affinity.

Three of the more potent AMO designs from the functional study performedin Example 3 were examined in greater detail to assess their relativeability to discriminate mismatches between the synthetic anti-miRNAoligonucleotide and their target. In general, high affinityoligonucleotides show high potency but usually show reduced specificityas the high affinity permits hybridization even in the presence of oneor more mismatches in complementarity. The designs “2′OMe 5′+3′iFQ”,“DNA/LNA-PS”, and “2′OMe/LNA-PS” were synthesized as variants having 1,2, or 3 mismatches to the miR-21 target sequence. Sequences are shown inTable 7. Studies were performed as described in Example 3.

TABLE 7  SEQ ID NO: Name Sequence 35 2′OMe 5′ + 3′iFQU_(z)C A A C A U C A G U C  U G A U A A G C U_(z)A 42 2′OMe 5′ + 3′iFQU_(z)C A A C A U C A G U C  1MUT U 

 A U A A G C U_(z)A 43 2′OMe 5′ + 3′iFQ U_(z)C A A 

 A U C A G U C  2MUT U 

 A U A A G C U_(z)A 44 2′OMe 5′ + 3′iFQ U_(z)C A A 

 A U C A G U C  3MUT U 

 A U A A G 

 U_(z)A 40 DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c* t*G*a*t*A*a*g*C*t*a 45DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c* 1MUT t*

*a*t*A*a*g*C*t*a 46 DNA/LNA PS t*C*a*a*

*a*t*C*a*g*T*c* 2MUT t*

*a*t*A*a*g*C*t*a 47 DNA/LNA PS t*C*a*a*

*a*t*C*a*g*T*c* 3MUT t*

*a*t*A*a*g*

*t*a 41 2′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C* U*G*A*U*A*A*G*C*U*A 482′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C* 1MUT U*

*A*U*A*A*G*C*U*A 49 2′OMe/LNA PS U*C*A*A*

*A*U*C*A*G*T*C* 2MUT U*

*A*U*A*A*G*C*U*A 50 2′OMe/LNA PS U*C*A*A*

*A*U*C*A*G*T*C* 3MUT U*

*A*U*A*A*G*

*U*A Uppercase = 2′OMe RNA Lowercase = DNA Uppercase with underscore =LNA “*” = phosphorothioate linkage “z” = napthylene-azo modifier (iFQ)Mutations are identified with bold italic font

Results. The RLuc/FLuc ratios obtained from transfections done with thevarious AMOs are shown in FIG. 3. In the untreated state, HeLa cellscontain large amounts of miRNA 21 that suppress expression of the RLucreporter. Any treatment that decreases miR-21 levels leads to anincrease in RLuc expression and thus increases the relative RLuc/FLucratio (with FLuc serving as an internal normalization control fortransfection efficiency). For each of the chemistries studied, theparent wild-type sequence is followed by variants having 1, 2, or 3mutations.

In all cases, the perfect match AMO showed significant suppression ofmiR-21 activity as evidenced by increases in luciferase levels (increasein the RLuc to FLuc ratio indicating de-repression of the RLuc mRNA). Asin Example 3 (FIG. 3), the “2′OMe/LNA-PS” compound showed the highestpotency as evidenced by suppression of miR-21 at low dose (1 nM and 5 nMdata points). The “2′OMe 5′+3′iFQ” and “DNA/LNA-PS” AMOs showedrelatively similar performance both in wild-type (perfect match) andmutant (mismatch) versions. In both cases, a single mismatch showed apartial reduction of anti-miR-21 activity, the double mismatch showedalmost complete elimination of anti-miR-21 activity, and the triplemismatch did not show any anti-miR-21 activity. In contrast, the higheraffinity “2′OMe/LNA-PS” compound showed significant anti-miR-21 activityfor both the single and double mismatch compounds and even showed someactivity at high dose (50 nM) for the triple mismatch compound. Thus,while the “2′OMe/LNA-PS” compound is most potent, it is also the leastspecific of the reagents studied.

Of note, the above experiments were performed using AMOs that placed themismatches at positions that are LNA modified (in the LNA containingAMOs). This design may influence the likelihood that a mismatch willaffect activity as it disrupts a high affinity LNA:RNA base pair. Thus,these results represent the best case scenario for specificity of theLNA-modified AMOs. The experiment was repeated using a new set ofreagents where the mismatches were all positioned at non-LNA bases. Thisnew series of AMO reagents is shown in Table 8.

TABLE 8  Synthetic oligomers employed in Example 4 (miR21 AMOs) SEQ IDNO: Name Sequence 35 2′OMe 5′ + U_(z)C A A C A U C A G U C U G A  3′iFQU A A G C U_(z)A 51 2′OMe 5′ + U_(z)C A A C A U C A G U C 

 G A  3′1FQ 1MUT v2 U A A G C U_(z)A 52 2′OMe 5′ + U_(z)C A A C 

 U C A G U C 

 G A  3′iFQ 2MUT v2 U A A G C U_(z)A 53 2′OMe 5′ + U_(z)C A A C 

 U C A G U C 

 G A  3′iFQ 3MUT v2 U A A 

 C U_(z)A 40 DNA/LNA PS t*C*a*a*C*a*t*C*a*g*T*c*t*G*a* t*A*a*g*C*t*a 54DNA/LNA PS  t*C*a*a*C*a*t*C*

*g*T*c*a*G*a* 1MUT v2 t*A*a*g*C*t*a 55 DNA/LNA PS  t*C*a*a*C*

*t*C*

*g*T*c*a*G*a* 2MUT v2 t*A*a*g*C*t*a 56 DNA/LNA PS  t*C*a*a*C*

*t*C*

*g*T*c*a*G*a* 3MUT v2 t*A*a*

*C*t*a 41 2′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*U*G*A* U*A*A*G*C*U*A 572′OMe/LNA PS U*C*A*A*C*A*U*C*A*G*T*C*

*G*A* 1MUT v2 U*A*A*G*C*U*A 58 2′OMe/LNA PS U*C*A*A*C*

*U*C*A*G*T*C*

*G*A* 2MUT v2 U*A*A*G*C*U*A 59 2′OMe/LNA PS U*C*A*A*C*

*U*C*A*G*T*C*

*G*A* 3MUT v2 U*A*A*

*C*U*A Uppercase = 2′OMe RNA Lowercase = DNA Uppercase with underscore =LNA “*” = phosphorothioate linkage “z” = napthylene-azo modifier (iFQ)Mutations are identified with bold italic font

Results. The RLuc/FLuc ratios obtained from transfections done with thevarious AMOs are shown in FIG. 4. In the untreated state, HeLa cellscontain large amounts of miRNA 21 that suppress expression of the RLucreporter. Any treatment that decreases miR-21 levels leads to anincrease in RLuc expression and thus increases the relative RLuc/FLucratio (with FLuc serving as an internal normalization control fortransfection efficiency). For each of the chemistries studied, theparent wild-type sequence is followed by variants having 1, 2, or 3mutations.

The results were nearly identical to those obtained with the originalmutation mismatch placement (FIG. 3). In all cases, the perfect matchAMO showed significant suppression of miR-21 activity as evidenced byincreases in luciferase levels (increase in the RLuc to FLuc ratioindicating de-repression of the RLuc mRNA). As in Example 3 (FIG. 3),the “2′OMe/LNA-PS” compound showed the highest potency as evidenced bysuppression of miR-21 at low dose (1 nM and 5 nM data points). The“2′OMe 5′+3′iFQ” and “DNA/LNA-PS” AMOs showed relatively similarperformance both in wild-type (perfect match) and mutant (mismatch)versions. In both cases, a single mismatch showed a partial reduction ofanti-miR-21 activity, the double mismatch showed almost completeelimination of anti-miR-21 activity, and the triple mismatch did notshow any anti-miR-21 activity. In contrast, the higher affinity“2′OMe/LNA-PS” compound showed significant anti-miR-21 activity for boththe single and double mismatch compounds and even showed some activityat high dose (50 nM) for the triple mismatch compound. Thus, while the“2′OMe/LNA-PS” compound is most potent, it is also the least specific ofthe reagents studied.

EXAMPLE 5

This example demonstrates improved functional activity of internalnapthylene-azo-containing oligomers at reducing cellular mRNA levelswhen incorporated into RNase H active ASOs as compared to other relatedcompounds.

Oligonucleotides antisense in orientation to cellular messenger RNAs(mRNAs) will hybridize to the mRNA and form an RNA/DNA heteroduplex,which is a substrate for cellular RNase H. Degradation by RNase H leadsto a cut site in the mRNA and subsequently to total degradation of thatRNA species, thereby functionally lowering effective expression of thetargeted transcript and the protein it encodes. ASOs of this typerequire a domain containing at least 4 bases of DNA to be a substratefor RNase H, and maximal activity is not seen until 8-10 DNA bases arepresent. ASOs must be chemically modified to resist degradation by serumand cellular nucleases. Phosphorothioate (PS) modification of theinternucleotide linkages is compatible with RNase H activation, howevermost other nuclease resistant modifications prevent RNase H activity,including all 2′-modifications, such as 2′OMe RNA, LNA, MOE, etc. The PSmodification lowers binding affinity (T_(m)). In general, modificationsthat lower T_(m) decrease potency while modifications that increaseT_(m) improve potency. One strategy to improve potency of ASOs is toemploy a chimeric design where a low T_(m), RNase H activating domainmade of PS-modified DNA is flanked by end domains that contain2′-modified sugars which confer high binding affinity but are not RNaseH activating (“gapmer” design). One commonly employed strategy is toplace five 2′-modified bases at the 5′-end, ten PS-modified DNA bases inthe middle, and five 2′-modified bases at the 3′-end of the ASO (socalled “5-10-5” design). A modification that confers nucleaseresistance, increases binding affinity, and does not impair thereagent's ability to activate RNase H would be ideal. The presentexample demonstrates the utility of the internal napthylene-azo modifierto improve the nuclease stability and increase binding affinity of ASOs,enhancing their function as gene knockdown reagents.

Oligonucleotide synthesis and purification. DNA, 2′OMe RNA, and LNAcontaining oligonucleotides were synthesized using solid phasephosphoramidite chemistry, deprotected and desalted on NAP-5 columns(Amersham Pharmacia Biotech, Piscataway, N.J.) according to routinetechniques (Caruthers et al., 1992). The oligomers were purified usingreversed-phase high performance liquid chromatography (RP-HPLC). Thepurity of each oligomer was determined by capillary electrophoresis (CE)carried out on a Beckman P/ACE MDQ system (Beckman Coulter, Inc.,Fullerton, Calif.). All single-strand oligomers were at least 85% pure.Electrospray-ionization liquid chromatography mass spectrometry(ESI-LCMS) of the oligonucleotides was conducted using an Oligo HTCSsystem (Novatia, Princeton, N.J.), which consisted of ThermoFinniganTSQ7000, Xcalibur data system, ProMass data processing software, andParadigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.). Protocolsrecommended by the manufacturers were followed. Experimental molarmasses for all single-strand oligomers were within 1.5 g/mol of expectedmolar mass. These results confirm identity of the oligomers.

TABLE 9  Synthetic oligomers employed in Example 5(anti-HPRT AS0s) SEQID NO: Name Sequence 60 HPRT#1 DNAa t a g g a c t c c a g a t g t t t c c 61 HPRT#1 DNA 5′iFQa_(z)t a g g a c t c c a g a t g t t t c c 62 HPRT#1 DNA 3′iFQa t a g g a c t c c a g a t g t t t c_(z)c 63 HPRT#1 DNA 5′ + 3′iFQa_(z)t a g g a c t c c a g a t g t t t c_(z)c 64 HPRT#1 DNA 5′ + i +3′iFQ a_(z)t a g g a c t c c_(z)a g a t g t t t c_(z)c 65 HPRT#1 DNA PSa*t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*c*c 66 HPRT#1 DNA PS 5′iFQa_(z)t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*c*c 67 HPRT#1 DNA PS 3′iFQa*t*ag*g*a*c*t*c*c*a*g*a*t*g*t*t*t*c_(z)c 68 HPRT#1 DNA PS 5′ + 3′iFQa_(z)t*a*g*g*a*c*t*c*c*a*g*a*t*g*t*t*t*c_(z)c 69 HPRT#1 DNA PSa_(z)t*a*g*g*a*c*t*c*cza*g*a*t*g*t*t*t*c_(z)c 5′ + I + 3′iFQ 70HPRT#1 5-10-5 A U A G G a c t c c a g a t g U U U C C 71HPRT#1 5-10-5 2x iFQ A_(z)U A G G a c t c c a g a t g U U U C_(z)C 72HPRT#1 5-10-5 3x iFQ A_(z)U A G G a c t c c_(z)a g a t g U U U C_(z)C 73HPRT#1 5-10-5 PS A*U*A*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U*C*C 74HPRT#1 5-10-5 PS 2x iFQ A_(z)U*A*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U*C_(z)C 75HPRT#1 5-10-5 gapPS A U A G G a*c*t*c*c*a*g*a*t*g*U U U C C 76HPRT#1 5-10-5 gapPS 2x A U A G G a*c*t*c*c*a*g*a*t*g*U U U C C iFQ 77HPRT#1 5-10-5 gapPS 3x A U A G G a*c*t*c*c_(z)a*g*a*t*g*U U U C C iFQ 78HPRT#1 5-10-5 LNA PS A*U*A*G*G*a*c*t*c*c*a*g*a*t*g*U*U*U*C*C Uppercase =2′OMe RNA Lowercase = DNA Uppercase with underscore = LNA “*” =phosphorothioate linkage “z” = napthylene-azo modifier (iFQ)

HeLa cell culture, transfections, and RT-qPCR methods. HeLa cells weresplit into 48-well plates and were transfected the next day at ˜60%confluency in serum-free Dulbecco's Modified Eagle Medium (Invitrogen,Carlsbad, Calif.) using TriFECTin® (Integrated DNA Technologies,Coralville, Iowa) at a concentration of 2% (1 μL per 50 μL, OptiMEM® I)(Invitrogen, Carlsbad, Calif.) with ASOs at the indicatedconcentrations. All transfections were performed in triplicate. After 6hours, media was exchanged with Dulbecco's Modified Eagle Mediumcontaining 10% fetal bovine serum. RNA was prepared 24 hours aftertransfection using the SV96 Total RNA Isolation Kit (Promega, Madison,Wis.). cDNA was synthesized using 150 ng total RNA with SuperScript™-IIReverse Transcriptase (Invitrogen, Carlsbad, Calif.) per themanufacturer's instructions using both random hexamer and oligo-dTpriming. Transfection experiments were all performed a minimum of threetimes.

Quantitative real-time PCR was performed using 10 ng cDNA per 10 μLreaction with Immolase™ DNA Polymerase (Bioline, Randolph, Mass.), 200nM primers, and 200 nM probe. Hypoxanthine phosphoribosyltransferase 1(HPRT1) (GenBank Acc. No. NM_000194) specific primers were:

HPRT-For  (SEQ ID NO: 79) 5′ GACTTTGCTTTCCTTGGTCAGGCA, HPRT-Rev (SEQ ID NO: 80) 5′ GGCTTATATCCAACACTTCGTGGG,  and  probe HPRT-P(SEQ ID NO: 81) 5′ MAX-ATGGTCAAGGTCGCAAGCTTGCTGGT-IowaBlackFQ   (IBFQ)and were normalized to levels of an internal control gene, human acidicribosomal phosphoprotein PO (RPLPO) (GenBank Acc. No. NM_001002), whichwas measured in a multiplexed reaction using primers:

RPLPO-For (SEQ ID NO: 82) 5′ GGCGACCTGGAAGTCCAACT, RPLPO-Rev (SEQ ID NO: 83) 5′ CCATCAGCACCACAGCCTTC,  and  probe RPLPO-P (SEQ ID NO: 84) 5′ FAM-ATCTGCTGCATCTGCTTGGAGCCCA-IBFQ(Bieche et al., 2000, Clin. Cancer Res. 6(2): 452-59). Cyclingconditions employed were: 95° C. for 10 minutes followed by 40 cycles of2-step PCR with 95° C. for 15 seconds and 60° C. for 1 minute. PCR andfluorescence measurements were done using an ABI Prism™ 7900 SequenceDetector (Applied Biosystems Inc., Foster City, Calif.). All reactionswere performed in triplicate. Expression data were normalized. Copynumber standards were multiplexed using linearized cloned amplicons forboth the HPRT and RPLP0 assays. Unknowns were extrapolated againststandards to establish absolute quantitative measurements.

Results. ASOs were transfected into HeLa cells at 1 nM, 5 nM, and 20 nMconcentrations. RNA was prepared 24 hours post transfection, convertedto cDNA, and HPRT expression levels were measured using qPCR. Resultsare shown in FIG. 5 for the set of anti-HPRT ASOs made from DNA bases.Unmodified single-stranded DNA oligos are rapidly degraded byexonucleases and endonucleases. No knockdown of HPRT was observed usingthis design (HPRT DNA), presumably due to rapid degradation of theunprotected compound. ASOs with a single iFQ modification near the3′-end (HPRT DNA 3′iFQ), a single iFQ modification near the 5′-end (HPRTDNA 5′iFQ), two iFQ modifications inserted near both ends (HPRT DNA5′+3′iFQ), and three iFQ modifications inserted in the center and nearboth ends (HPRT DNA 5′+I+3′iFQ) were also tested and similarly showed nofunctional gene knockdown at the doses examined. Therefore, the additionof even up to 3 iFQ modifications does not provide sufficient nucleasestabilization to permit otherwise unmodified DNA oligos to function asantisense gene-knockdown agents.

The same series of oligonucleotides was synthesized havingphosphorothioate (PS) intemucleotide bonds throughout the sequence(except where the phosphate connects to an iFQ modifier). Historically,DNA-PS oligos were among the first effective antisense compoundsstudied. This modification increases nuclease stability; however, italso lowers binding affinity (T_(m)) and as a result this so-called“first generation” antisense chemistry usually shows relatively lowpotency. The “DNA-PS” ASO reduced HPRT levels by 50% at 20 nMconcentration; however, no reduction in HPRT levels was observed atlower doses. Addition of the iFQ modification, which increases bindingaffinity and blocks exonuclease action, improved function of the DNA-PSASOs. The “DNA-PS 5′+I+3′iFQ” compound showed the best results withinthis series, with HPRT knockdown of 70% at 20 nM and 40% at 5 nMobserved (FIG. 5).

A second set of ASOs was synthesized using a chimeric “5-10-5 gapmer”design where five base end domains were made of 2′OMe RNA and a centralten base RNase H active domain were made of DNA. Oligonucleotides hadzero, one, two, or three iFQ modifiers inserted at the same positions asthe DNA ASOs in FIG. 5. These oligonucleotides were transfected intoHeLa cells as before and HPRT mRNA levels were examined 24 hourspost-transfection. Results are shown in FIG. 6. The three gapmer ASOswith a phosphodiester DNA central domain showed no activity in reducingHPRT mRNA levels, regardless of whether the sequence was modified withthe iFQ group or not (“5-10-5”, 5-10-5 2× iFQ” and “5-10-5 3× iFQ”). Thesame sequence fully PS modified (“5-10-5-PS”) showed good potency with75% knockdown of HPRT at 20 nM concentration. The addition of two iFQgroups near the ends of this design (“5-10-5-PS 2× iFQ”) showed the bestpotency of this set, with >90% knockdown of HPRT at 20 nM and >70%knockdown at 5 nM concentration.

Although 2′OMe RNA is somewhat resistant to endonuclease attack, gapmerASOs of this design are usually made with full PS modification toprevent exonuclease degradation. Consistent with this idea, an ASO withthe DNA domain protected by PS internucleotide linkages but havingphosphodiester bonds in the 2′OMe flanking domains showed no geneknockdown activity (“5-10-5 gapPS”). Use of the iFQ modification at theends, however, permits use of this new design by providing protectionfrom exonuclease attack; this new design should also increase bindingaffinity and lower toxicity by reducing PS content. This strategy waseffective and the ASO (“5-10-5 gapPS 2× iFQ”) showed knockdown of HPRTlevels by >90% at 20 nM and by >70% at 5 nM. Potency was very similar tothe full PS modified ASO. This design is expected to have reducedtoxicity; however, toxicity is not easily tested in this system as HeLacells are tolerant to fairly high doses of PS modified oligonucleotides.Benefit from reduced PS content will be better appreciated in vivo.

Although it did not increase functional potency, addition of a thirdcentrally placed iFQ group (“5-10-5 gapPS-3×-iFQ”) was compatible withgene knockdown in this RNase H active antisense design. It is generallyaccepted that maximal activity of RNase H active ASOs requires a DNAdomain having at least 8 uninterrupted DNA residues. It was unexpectedthat the 3× iFQ design (where the 10 base DNA domain is interrupted by acentral iFQ group) would work without reducing potency compared with the2× iFQ design (where the 10 base DNA domain is continuous). It ispossible that unique properties of the iFQ group allow its insertion toremain compatible with RNase H activity, possibly due to the samepostulated base stacking interactions that result in increased T_(m) inthese compounds.

The most potent antisense design in current use are LNA-modifiedgapmers, where very strong T_(m) enhancing LNA modifications are used inthe flanking domains in place of the 2′OMe RNA bases used in the presentexample. While potent, this design is expensive and can show significanttoxicity in certain contexts. The same anti-HPRT sequence was made as anLNA 5-10-5 gapmer (fully PS modified). As expected, this compound showedthe highest relative potency of any of the ASOs tested (“5-10-5 LNA PS”)but the observed potency was only marginally higher than the best of theiFQ compositions (“5-10-5-PS 2× iFQ”). The very high binding affinityLNA reagents usually result in decreased specificity, so use of the iFQdesigns of the present invention may show improved specificity at asmall cost in potency.

EXAMPLE 6

This example demonstrates use of the iFQ modification in RNA duplexeswith application in suppressing gene expression via an RNAi mechanism ofaction.

The use of double-stranded RNA (dsRNA) to trigger gene suppression viaRNA interference (RNAi) is a well-described technique. Synthetic dsRNAsthat mimic natural cellular products (small interfering RNAs, or siRNAs)are usually 21 bases long with a central 19 base duplex domain with2-base 3′-overhangs. Alternatively, slightly larger syntheticoligonucleotides can be used that are substrates for the cytoplasmicnuclease Dicer, which processes these species into 21-mer siRNAs.Typically these reagents are asymmetric and have a 25 base top (Sensestrand, “S”) and a 27 base bottom strand (Antisense strand, “AS”) with asingle 2-base 3′-overhang on the AS strand. These longer siRNAs arecalled Dicer-substrate siRNAs, or DsiRNAs. Although dsRNA is far morestable to nuclease attack than single-stranded RNA (ssRNA), degradationof the synthetic siRNAs can significantly limit potency of thecompounds, especially when used in vivo. Incorporation of chemicalmodifications, such as 2′OMe RNA, 2′F RNA, or LNA bases, improvesnuclease stability and can improve function of the siRNA. Selectiveplacement of nuclease-resistant phosphorothioate bonds (PS) can alsohelp stabilize the siRNA, especially when used near the terminal3′-internucleotide linkages. Unfortunately, careful placement ofmodified groups is essential as extensive chemical modification usuallylowers functional potency of the compound even though nucleasestabilization has been achieved, probably through disrupting interactionof the RNA duplex with key protein mediators of RNAi, like Dicer orAgo2.

The present example demonstrates that the iFQ modifier can be introducedinto DsiRNAs. Like other chemical modifiers, iFQ insertion can lead toincreased potency, decreased potency, or no change in potency dependingupon placement.

Oligonucleotide synthesis and purification. RNA and modified RNAoligonucleotides were synthesized using solid phase phosphoramiditechemistry, deprotected and desalted according to routine techniques(Caruthers et al., 1992). The oligomers were purified using ion-exchangehigh performance liquid chromatography (IE-HPLC) and were handled underRNase-free conditions. All RNA oligonucleotides were prepared as asodium salt. The purity of each oligomer was determined by capillaryelectrophoresis (CE) carried out on a Beckman P/ACE MDQ system (BeckmanCoulter, Inc., Fullerton, Calif.). All single-strand oligomers were atleast 85% pure. Electrospray-ionization liquid chromatography massspectrometry (ESI-LCMS) of the oligonucleotides was conducted using anOligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.

Duplexes were formed by mixing equal molar amounts of the top and bottomstrands in 30 mM Hepes, pH 7.5, 100 mM potassium acetate, heating at 95°C. for 2 minutes, then cooling to room temperature. Table 10 lists theduplexes synthesized for Example 6.

TABLE 10  Synthetic RNA duplexes employed in Example 6(anti-HPRT DsiRNAs) SEQ ID NO: Name Sequence 85 NCI   5′  CGUUAAUCGCGUAUAAUACGCGUat 3′ S 86 Negative 3′CAGCAAUUAGCGCAUAUUAUGCGCAUA 5′ AS Control 87 HPRT 5′  GCCAGACUUUGUUGGAUUUGAAAtt 3′ S 88 unmod 3′UUCGGUCUGAAACAACCUAAACUUUAA 5′ AS 89 HPRT  5′   GCCAGACUUUGUUGGAUUUGAAAtt 3′ S 90 iFQ v1 3′U^(z)UCGGUCUGAAACAACCUAAACUUUAA 5′ AS 91 HPRT  5′  G^(z)CCAGACUUUGUUGGAUUUGAAAtt 3′ S 92 iFQ v2 3′UUC GGUCUGAAACAACCUAAACUUUAA 5′ AS 93 HPRT  5′  G^(z)CCAGACUUUGUUGGAUUUGAAAt^(z)t 3′ S 94 iFQ v3 3′UUCGGUCUGAAACAACCUAAACUUUA A 5′ AS 95 HPRT  5′  G CCAGACUUUGUUGGAUUUGAAAtt 3′ S 96 iFQ v4 3′UUC^(z)GGUCUGAAACAACCUAAACUUUAA 5′ AS 97 HPRT  5′  G CCAGACUUUGUUGGAUUUGAAAt t 3′ S 98 iFQ v5 3′UUC^(z)GGUCUGAAACAACCUAAACUUUA^(z)A 5′ AS 99 HPRT  5′  G^(z)CCAGACUUUGUUGGAUUUGAAAt^(z)t 3′ S 100 iFQ v6 3′UUC^(z)GGUCUGAAACAACCUAAACUUUA A 5′ AS 101 HPRT  5′  G^(z)CCAGACUUUGUUGGAUUUGAAAt^(z)t 3′ S 102 iFQ v7 3′UUC^(z)GGUCUGAAACAACCUAAACUUUA^(z)A 5′ AS Uppercase = RNA Lowercase =DNA “z” = insertion of napthylene-azo modifier (iFQ) Note: gaps havebeen introduced in sequences for the purpose of alignment only and donot represent any modification to sequence.

HeLa cell culture, transfections, and RT-qPCR methods. HeLa cells weretransfected in “reverse format” at ˜60% confluency (Invitrogen,Carlsbad, Calif.) using 1 Lipofectamine™ RNAiMAX per 50 μL OptiMEM™ I(Invitrogen, Carlsbad, Calif.) with RNA duplexes at the indicatedconcentrations. All transfections were performed in triplicate. RNA wasprepared 24 hours after transfection using the SV96 Total RNA IsolationKit (Promega, Madison, Wis.); cDNA was synthesized using 150 ng totalRNA with SuperScript™-II Reverse Transcriptase (Invitrogen, Carlsbad,Calif.) per the manufacturer's instructions using both random hexamerand oligo-dT priming.

Quantitative real-time PCR reactions were done using 10 ng cDNA per 10μL reaction, Immolase™ DNA Polymerase (Bioline, Randolph, Mass.), 500 nMprimers, and 250 nM probe. Hypoxanthine phosphoribosyltransferase 1(HPRT1) (GenBank Acc, No. NM_000194) specific primers were:

HPRT-For (SEQ ID NO: 79) 5′ GACTTTGCTTTCCTTGGTCAGGCA, HPRT-Rev(SEQ ID NO: 80) 5′ GGCTTATATCCAACACTTCGTGGG,  and  probe HPRT-P (SEQ ID NO: 81) 5′ FAM-ATGGTCAAGGTCGCAAGCTTGCTGGT-IowaBlackFQ. (IBFQ)Cycling conditions employed were: 95° C. for 10 minutes followed by 40cycles of 2-step PCR with 95° C. for 15 seconds and 60° C. for 1 minute.PCR and fluorescence measurements were done using an ABI Prism™ 7900Sequence Detector (Applied Biosystems Inc., Foster City, Calif.). Alldata points were performed in triplicate. Expression data werenormalized to levels of an internal control gene, human splicing factor,arginine/serine-rich 9 (SFRS9) (GenBank Acc. No. NM_003769), which wasmeasured in a multiplexed reaction using primers:

SFRS9-For  (SEQ ID NO: 103) 5′ TGTGCAGAAGGATGGAGT, SFRS9-Rev (SEQ ID NO: 104) 5′ CTGGTGCTTCTCTCAGGATA, and  probe SFRS9-P (SEQ ID NO: 105) 5′ MAX-TGGAATATGCCCTGCGTAAACTGGA-IBFQ,the baseline to cells transfected with a scrambled negative control RNAduplex (NCl). Copy number standards were run in parallel usinglinearized cloned amplicons for both the HPRT and SFRS9 assays. Unknownswere extrapolated against standards to establish absolute quantitativemeasurements.

Results. The anti-HPRT DsiRNA employed in the present study is extremelypotent and typically shows detectable knockdown of target mRNA at lowpicomolar levels. Consistent with this expectation, the unmodifiedduplex reduced HPRT levels by ˜40% at a 10 pM dose at 24 hourspost-transfection in HeLa cells. A series of modified duplexescontaining the iFQ group positioned at various locations in the Sstrand, AS strand, or both were similarly transfected into HeLa cellsand HPRT mRNA levels were measured 24 hour post-transfection. Resultsare shown in FIG. 7.

Placing the iFQ group near the 3′-end of the AS strand was welltolerated; insertion between bases 1 and 2 from the 3′-end (in thesingle-stranded 3′-overhang domain) (duplex HPRT iFQ v1) or betweenbases 3 and 4 from the 3′-end (at the start of the duplex domain)(duplex HPRT iFQ v4) showed similar potency to the unmodified duplex.Placing the iFQ group near the 5′-end of the S strand was similarly welltolerated (duplex HPRT iFQ v2) as was placing the iFQ group near bothends of the S strand (duplex HPRT iFQ v3). In contrast, duplexes havingan iFQ group near the 5′-end of the AS strand showed reduced potency(duplexes HPRT iFQ v5 and v7), so modification at this position shouldbe avoided.

Within the error of the system studied, the iFQ modified and unmodifiedduplexes showed similar potency (except for those duplexes modified atthe 5′-end of the AS strand, as noted above). Benefit from the iFQ groupis most likely to be-evident in settings where nuclease stabilization isneeded, which is not appreciated in the present in vitro system, butbased on the results of Examples 1 and 2, greater benefit would beexpected from use of this modification when used in vivo where exposureto serum nucleases is more problematic.

EXAMPLE 7

This example demonstrates decreased cellular toxicity from lipidtransfected internal napthylene-azo-containing oligomers compared withother compounds.

Toxicity from chemical modification of synthetic oligomers can beproblematic as it can give unwanted side effects, cause unreliableresults, and limit therapeutic utility of the oligomer. Cellular deathcan result from toxic chemical modifications by inducing necrosis orapoptosis. Toxicity was ascertained with oligomers containing anon-targeting, negative control (“NCl”) sequence using chemicalmodification patterns employed in the AMOs examined in Examples 3 and 4(see Table 11). Generalized cytotoxicity (from necrosis and/orapoptosis) was measured by quantifying the relative number of live anddead cells after treatment with the chemically modified oligomers, whilecytotoxicity resulting from the induction of the apoptotic pathway wasdetermined by measuring the levels of caspase-3 and -7 after oligomertreatment.

Oligonucleotide synthesis and preparation. DNA oligonucleotides weresynthesized using solid phase phosphoramidite chemistry, deprotected anddesalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.)according to routine techniques (Caruthers et al., 1992). The oligomerswere purified using reversed-phase high performance liquidchromatography (RP-HPLC). The purity of each oligomer was determined bycapillary electrophoresis (CE) carried out on a Beckman P/ACE MDQ system(Beckman Coulter, Inc., Fullerton, Calif.). All single-strand oligomerswere at least 90% pure. Electrospray-ionization liquid chromatographymass spectrometry (ESI-LCMS) of the oligonucleotides was conducted usingan Oligo HTCS system (Novatia, Princeton, N.J.), which consisted ofThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processingsoftware, and Paradigm MS4™ HPLC (Michrom BioResources, Auburn, Calif.).Protocols recommended by the manufacturers were followed. Experimentalmolar masses for all single-strand oligomers were within 1.5 g/mol ofexpected molar mass. These results confirm identity of the oligomers.

TABLE 11  Synthetic oligomers employed in Example 7 (NC1 AMOs) SEQ IDNO: Name Sequence 106 2′OMe 5′ + 3′ iFQ  G_(z)C G U A U U A U A G C C G A U U A A C G_(z)A 107 2′OMe PSends G*C*G*U A U U A U A G C C G A U U A A*C*G*A 108 DNA/PS g*c*g*t*a*t*t*a*t*a*g*c*c*g* a*t*t*a*a*c*g*a109 DNA/LNA PO g C g t A t t A t a G c c G  a t T a a C g a 110DNA/LNA PS g*C*g*t*A*t*t*A*t*a*G*c*c*G* a*t*T*a*a*C*g*a 111 2′OMe/LNA POG C G T A T T A T A G C C G  A T T A A C G A 112 2′OMe/LNA PSG*C*G*T*A*T*T*A*T*A*G*C*C*G* A*T*T*A*A*C*G*A Uppercase = 2′OMe RNALowercase = DNA Uppercase with underscore = LNA “*” = phosphorothioatelinkage “z” = napthylene-azo modifier (iFQ)

Cell culture, transfections, and luciferase assays. HeLa cells wereplated in 48-well plates in DMEM containing 10% FBS to achieve 90%confluency the next day. The following morning, NCl AMOs weretransfected at 100 nM or 50 nM concentrations in triplicate wells in twosets (one for measuring general cytotoxicity, one for measuringapoptosis induction) with 1 μl TriFECTin® (Integrated DNA Technologies)per well in DMEM containing 10% FBS. An apoptosis-inducing agent,Staurosporine (1 mM in DMSO), was incubated at 1 μM on the cells for 24hours as a positive control. After 24 hours of NCl AMO treatment, thefirst set of cells was analyzed for viability using the MultiTox-GloMultiplex Cytotoxicity Assay (Promega, Madison, Wis.) with thepeptide-substrate GF-AFC (glycyl-phenylalanylaminofluorocoumarin), whichgenerates a fluorescence signal upon cleavage by a “live-cell” specificprotease, measured at 405 nm_(Ex)/505 nm_(Em) in a SpectraFluorMicroplate Reader (Tecan Group Ltd, Männedorf, Switzerland). Continuingto use the MultiTox-Glo Multiplex Cytotoxicity Assay, the same cellswere subsequently analyzed for cytotoxicity by detecting a “dead-cell”protease activity in a luciferase-based assay measured on a GloMax® 96Microplate Luminometer (Promega) per the manufacturer's recommendations.To assess cytotoxicity derived from induction of the apoptosis pathway,the Caspase-Glo® 3/7 Assay (Promega) was performed with the second setof cells to measure caspase-3 and -7 levels according to themanufacturer's recommendations on a GloMax® 96 Microplate Luminometer(Promega).

Results. For the cytoxocity analysis graphed in FIG. 8, data ispresented as a ratio of live/dead cells as calculated from the abundanceof “live-cell” and “dead-cell” proteases described above. The ratio oflive/dead cells serves as an internal normalizing control providing dataindependent of cell number, and a reduction of live/dead cellscorrelates with cytotoxicity. The “2′OMe 5′+3′ iFQ” and “2′OMe PSends”compounds are the least toxic oligomers and there is minimal toxicityeven at the high 100 nM dose. The “DNA/PS” oligomer, which is entirelycomprised of PS linkages, shows substantial cell death at both dosessuggesting that PS modification is toxic to the cells. When LNA basesare incorporated into the NC! AMO, such as in the “DNA/LNA PO” oligomer,cell death is seen at the high 100 nM dose suggesting that LNAmodification is toxic to the cells. Importantly, additive cell death isseen after combining these two chemistries in the “DNA/LNA PS” oligomer,demonstrating toxicity which also correlates with the dysmorphic,unhealthy cells seen during the visual analysis at the time the assaywas performed. Substituting DNA with 2′OMe bases in the “2′OMe/LNA PO”and “2′OMe/LNA PS” NCl AMOs reduces toxicity compared with their DNAcounterparts; however, cell death is still seen at the 100 nM dose.

In parallel, HeLa cells treated with the NCl AMOs were assessed forapoptosis induction by evaluating the levels of caspase-3 and -7 in aluciferase-based assay (FIG. 9). Luminescence is proportional to theabundance of the apoptosis effectors and an increase in RLUs correlateswith an increase in apoptosis. The data in FIG. 9 mirrors thecytotoxicity profiles from the NCI AMOs assayed in FIG. 8. The NCI AMOsthat do not trigger apoptosis are the “2′OMe 5′+3′ iFQ” and “2′OMePSends” compounds. Both extensive PS modification (“DNA/PS”) andincorporation of LNA bases (“DNA/LNA PO”) induce apoptosis, while anadditive effect is seen when these two chemistries are combined(“DNA/LNA PS”). Again, substitution of DNA bases for 2′OMe bases(“2′OMe/LNA PO” and “2′OMe/LNA PS”) reduces apoptosis induction.However, the “2′OMe/LNA PS” still demonstrates apoptosis induction atthe 100 nM dose.

This cytotoxicity profiling analysis clearly exemplifies that certainchemical modification strategies can be detrimental to cell viability.The “2′OMe 5′+3′ iFQ” AMO and the “DNA/LNA PS” AMOs, which demonstratedsimilar high potency in Example 3, have significantly different toxicityprofiles. The “2′OMe 5′+3′ iFQ” oligomer was non-toxic in this system,and the “DNA/LNA PS” oligomer caused substantial cell death in FIG. 8and was shown to induce apoptosis in FIG. 9. These data confirm thesuperiority of the “2′OMe 5′+3′ iFQ” AMO when compared to other standardAMOs with comparable potency (Example 3), increased specificity (Example4), and reduced toxicity.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of detecting a target oligonucleotide in a sample, themethod comprising: (a) contacting the sample with a compositioncomprising a probe oligonucleotide capable of hybridizing with thetarget oligonucleotide, the probe oligonucleotide having the structure5′-Y₁-X-Y₂-3′, wherein Y₁ comprises a fluorophore at or near the5′-terminus of the probe oligonucleotide and a sequence of 8-12 DNA orRNA nucleotides comprising a nucleotide N₁ having a 3′ phosphatecovalently linked to X; Y₂ comprises a quencher located at or near the3′-terminus of the probe oligonucleotide and a sequence of DNA or RNAnucleotides comprising a nucleotide N₂ having a 5′ phosphate covalentlylinked to X; and X comprises an internal quencher; wherein fluorescenceof the fluorophore is reduced when the probe oligonucleotide is nothybridized to the target oligonucleotide; and (b) detecting the presenceof the target oligonucleotide in the sample when an increase influorescence of the composition is detected as compared to thefluorescence of the composition in a control sample devoid of the targetoligonucleotide.
 2. The method of claim 1, wherein the quencher locatedat or near the 3′-terminus of the probe oligonucleotide is dabcyl,Eclipse® quencher, Black Hole quencher BHQ1, Black Hole quencher BHQ2,Black Hole quencher BHQ3, Iowa Black® FQ, Iowa Black® RQ-n1, or IowaBlack® RQ-n2.
 3. The method of claim 1, wherein the internal quencher isdabcyl, Eclipse® quencher, Black Hole quencher BHQ1, Black Hole quencherBHQ2, Black Hole quencher BHQ3, Iowa Black® FQ, Iowa Black® RQ-nl, orIowa Black® RQ-n2.
 4. The method of claim 1, wherein fluorescence of thefluorophore is reduced by fluorescence resonance energy transfer, groundstate quenching, or a combination thereof when the probe oligonucleotideis not hybridized to the target oligonucleotide.
 5. The method of claim1, wherein the increase in fluorescence arises from cleavage of theprobe oligonucleotide.
 6. The method of claim 1, wherein the probeoligonucleotide forms a random-coil conformation when the probeoligonucleotide is unhybridized, such that the fluorescence of thefluorophore is reduced.
 7. The method of claim 1, wherein the probeoligonucleotide comprises a self-complementary sequence and wherein thequencher and the fluorophore are attached to the probe oligonucleotidesuch that the fluorescence of the fluorophore is quenched when the probeoligonucleotide undergoes intramolecular base pairing.
 8. The method ofclaim 1, wherein the method is used in a polymerase chain reaction(PCR), wherein synthesis of PCR product results in an increase influorescence.
 9. The method of claim 1 wherein the stability of anoligonucleotide duplex comprising the probe oligonucleotide is greaterthan the stability of an oligonucleotide duplex comprising a comparatorprobe oligonucleotide lacking an internal quencher.
 10. The method ofclaim 1, wherein Y₁ comprises a fluorophore at the 5′-terminus of theprobe oligonucleotide and a sequence of 9 DNA or RNA nucleotidescomprising a nucleotide N₁ having a 3′ phosphate covalently linked to X.